Method for identifying time of bolting of chinese cabbage at seedling stage

Abstract

The application discloses a method for identifying the bolting time of Chinese cabbage at the seedling stage, and belongs to the technical field of molecular biology. The method uses the genomic DNA of Chinese cabbage as a template, adopts primer group 6943bp-insert-2F / 2R to amplify the fragment through a polymerase chain reaction, and according to the size of the target band, early bolting materials and varieties of Chinese cabbage can be quickly identified. The identification method provided by the application can detect early bolting materials and varieties of Chinese cabbage at an early development stage, improves the detection efficiency, saves a large amount of manpower and material resources, and further speeds up the breeding process of Chinese cabbage.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a method for identifying the bolting time of non-heading Chinese cabbage during the seedling stage. Background Technology

[0002] Non-heading cabbage ( Brassica campestris (syn.) Brassica rapa )ssp chinensis Originating in my country, non-heading Chinese cabbage is a subspecies of the genus *Brassica* in the family Brassicaceae. It is mainly cultivated in the middle and lower reaches of the Yangtze River and is popular with consumers due to its rich content of soluble sugars, organic acids, and trace elements essential for the human body. Different varieties or materials of non-heading Chinese cabbage exhibit differences in bolting time, resulting in variations in their vegetative growth. Early bolting of non-heading Chinese cabbage leads to a decline in both quality and yield. Therefore, research on bolting in non-heading Chinese cabbage is of significant guiding importance for improving its economic value and accelerating breeding.

[0003] Therefore, designing a method specifically for identifying premature bolting in non-heading Chinese cabbage during the seedling stage is extremely crucial. Summary of the Invention

[0004] The purpose of this invention is to provide a method for identifying the bolting time of non-heading Chinese cabbage during the seedling stage.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: Application of primer set 6943bp-insert-2F / 2R in seedling identification of early bolting materials or varieties of non-heading Chinese cabbage; The sequence of the primer set 6943bp-insert-2F / 2R is as follows: 6943bp-insert-2F:5'-GGCTATGTGCTCCCACCTTC -3'(Seq_1) 6943bp-insert-2R: 5'-CTAAATCGGTTAGTACTAGC-3'(Seq_2).

[0006] A method for identifying early bolting materials or varieties of non-heading Chinese cabbage during the seedling stage includes the following steps: Step 1: Take tissue from the material or variety to be identified and extract genomic DNA; Step 2: Using the genomic DNA extracted in Step 1 as a template, PCR amplification was performed on it using primer set 6943bp-insert-2F / 2R; The 6943bp insert-2F / 2R primer sequence is as follows: 6943bp-insert-2F: 5'-GGCTATGTGCTCCCACCTTC-3' 6943bp-insert-2R: 5'-CTAAATCGGTTAGTACTAGC -3'; Step 3: Separate the amplification products from Step 2 by agarose gel electrophoresis to obtain the band pattern of the material or variety to be identified. If the fragment size is 1687bp, it indicates that the material or variety exhibits early bolting characteristics.

[0007] Further, in step 2, the PCR reaction system is as follows: in a 10 μL reaction system, there is 3 μL of deionized water, 0.5 μL each of 10 μM 6943bp-insert-2F / 2R primers, 1 μL of 80 ng / μL genomic DNA, and 5 μL of 2× PrimeSTAR MAX DNAPolymerase.

[0008] Furthermore, in step 2, the PCR amplification reaction program is as follows: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and storage at 10℃.

[0009] Beneficial effects: 1. This invention enables rapid screening of early bolting varieties and materials of non-heading Chinese cabbage during the seedling stage by using the marker primer 6943bp-insert-2F / 2R.

[0010] 2. This invention provides convenient, low-cost, and simple selection assistance. Conventional breeding methods for the bolting trait in non-heading Chinese cabbage are time-consuming and labor-intensive. This invention allows selection to be performed during the seedling stage, significantly reducing subsequent screening costs. Attached Figure Description

[0011] Figure 1 , Figure 2 The image shows the agarose gel electrophoresis results of primer 6943bp-insert-2F / 2R amplified in various non-heading Chinese cabbage samples. Where M is the DL2000 Marker. Figure 1 The middle lanes 1-32, 35, and 40 are early bolting varieties, while lanes 33 and 36-39 are late bolting varieties. Figure 2 The varieties in lanes 1-34 are all early bolting varieties. Detailed Implementation

[0012] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used are commercially available, and techniques not described in detail are performed according to standard methods well known to those skilled in the art. The reagents mentioned in this application are commercially available or otherwise publicly available; they are merely examples and not the only ones applicable to the present invention. Other suitable tools or biological materials can be substituted. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0013] This invention provides a set of molecular marker primers, 6943bp-insert-2F / 2R, for identifying early bolting in non-heading Chinese cabbage during the seedling stage. Using genomic DNA from multiple early and late bolting varieties and materials of non-heading Chinese cabbage as templates, the target fragment is amplified via polymerase chain reaction. Based on the size of the target band, early bolting varieties and materials can be screened. This method provides a pair of molecular markers for detecting early bolting in non-heading Chinese cabbage during the seedling stage, enabling efficient and rapid detection of early bolting materials and varieties, saving breeding time and costs, thereby accelerating the breeding process and promoting the vigorous development of leafy vegetables.

[0014] Example 1 This implementation provides a method for identifying early bolting in non-heading Chinese cabbage during the seedling stage, including the following steps: Step 1: Extraction of genomic DNA from non-heading Chinese cabbage DNA was extracted according to the instructions of the Plant Genomic DNA Rapid Extraction Kit (purchased from Beijing Tiangen Biotech Co., Ltd.), as follows: 1) Take 100mg of leaves from non-heading Chinese cabbage material or variety, put them in liquid nitrogen and grind them thoroughly; 2) Before the experiment, preheat the centrifuge tube containing 700µL of GP1 in a 65℃ water bath, then add mercaptoethanol to bring the final concentration of GP1 to 0.1%. Add the above GP1 to the submerged powder, mix quickly, and place it in a 65℃ water bath for 20 min; 3) Add 700µL of chloroform to the above reaction solution and mix thoroughly. Centrifuge at 10,000rpm for 5min. 4) Transfer the supernatant obtained from the above centrifugation to a new centrifuge tube, add 700µL of GP2 buffer and mix well; 5) Transfer the above-mixed liquid to the adsorption column CB3, centrifuge at 10,000 rpm for 30 s, and discard the waste liquid; 6) Add 500µL of buffer GD to the adsorption column CB3, centrifuge at 10,000rpm for 30s, and discard the waste liquid; 7) Add 600µL of washing solution PW to the adsorption column CB3 again, centrifuge at 10,000rpm for 30s, discard the waste liquid, and put the adsorption column CB3 into the collection tube. 8) Repeat step 7; 9) Place the adsorption column CB3 back into the collection tube, centrifuge at 10,000 rpm for 2 minutes, and discard the waste liquid. Open the cap of the adsorption column CB3 and let it stand at room temperature for 2 minutes to thoroughly remove the washing liquid from the adsorption material; 10) Place the adsorption column CB3 into a new centrifuge tube, add 50 μL of elution buffer TE to the middle of the adsorption column CB3, incubate at room temperature for 2 min, centrifuge at 10,000 rpm for 2 min, and collect the genomic DNA into the centrifuge tube. 11) Determine the purity and concentration of the DNA, dilute it to 80 ng / μL, and store it in a -20°C freezer.

[0015] Step 2: Amplify the genomic DNA of non-heading Chinese cabbage using primer 6943bp-insert-2F / 2R. In a 10 μL reaction system, there were 1 μL of 80 ng / μL DNA template, 0.5 μL each of 10 μM labeled primers 6943 bp-insert-2F / 2R, 5 μL of 2× PrimeSTAR MAX DNA Polymerase, and 3 μL of deionized water.

[0016] 6943bp insert-2F / 2R labeled primers: 6943bp-insert-2F: 5'-GGCTATGTGCTCCCACCTTC-3', 6943bp-insert-2R: 5'-CTAAATCGGTTAGTACTAGC-3'.

[0017] The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and storage at 10℃.

[0018] Step 3, Agarose gel electrophoresis detection After PCR amplification, 1% agarose gel electrophoresis was performed using 1×TAE buffer. 10 μL of the PCR product was loaded onto the gel and electrophoresed at 150V for 15 min. The gel was then observed using a gel imaging system.

[0019] The electrophoresis results show that, Figure 1Early bolting varieties in lanes 1-32, 35, and 40 could all amplify a 1687bp fragment, while lanes 4, 33, and 36 (the fragment in lane 4 was relatively faint, but it was confirmed that a 1687bp fragment was present) and late bolting varieties in lanes 33 and 36-39 could not amplify a 1687bp fragment. Figure 2 Early bolting varieties in lanes 1-34 were all able to amplify a 1687bp fragment.

[0020] The results above show that when the molecular marker primer 6943bp-insert-2F / 2R is used to amplify the genome of non-heading Chinese cabbage, if a fragment of 1687p is amplified, it indicates that the non-heading Chinese cabbage variety or material has the trait of early bolting.

Claims

1. Application of primer set 6943bp-insert-2F / 2R in the identification of early bolting materials or varieties of non-heading Chinese cabbage during the seedling stage. The sequence of the primer set 6943bp-insert-2F / 2R is as follows: 6943bp-insert-2F:5'-GGCTATGTGCTCCCACCTTC-3' 6943bp-insert-2R: 5'-CTAAATCGGTTAGTACTAGC-3'; Using the genomic DNA of non-heading Chinese cabbage from early and late bolting varieties / materials as templates, the target fragment was amplified by polymerase chain reaction. Based on the size of the target band, early bolting varieties / materials were screened. Early bolting varieties could amplify a 1687bp fragment, while late bolting varieties could not amplify a 1687bp fragment.

2. A method for identifying early bolting materials or varieties of non-heading Chinese cabbage during the seedling stage, characterized in that: Includes the following steps: Step 1: Take tissue from the material or variety to be identified and extract genomic DNA; Step 2: Using the genomic DNA extracted in Step 1 as a template, PCR amplification was performed on it using primer set 6943bp-insert-2F / 2R; The 6943bp insert-2F / 2R primer sequence is as follows: 6943bp-insert-2F:5'-GGCTATGTGCTCCCACCTTC-3' 6943bp-insert-2R: 5'-CTAAATCGGTTAGTACTAGC-3'; Step 3: Separate the amplification products from Step 2 by agarose gel electrophoresis to obtain the band pattern of the material or variety to be identified. If the fragment size is 1687bp, it indicates that the material or variety has an early bolting trait; late bolting varieties cannot amplify a 1687bp fragment.

3. The method according to claim 2, characterized in that: In step 2, the PCR reaction system is as follows: in a 10 μL reaction system, there is 3 μL of deionized water, 0.5 μL each of 10 μM 6943bp-insert-2F / 2R primers, 1 μL of 80 ng / μL genomic DNA, and 5 μL of 2× PrimeSTAR MAX DNA Polymerase.

4. The method according to claim 2, characterized in that: In step 2, the PCR amplification reaction program is as follows: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles, and storage at 10℃.

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